CN105012333B - A kind of efficient anti-inflammatory PB ODN sequence and its application - Google Patents
A kind of efficient anti-inflammatory PB ODN sequence and its application Download PDFInfo
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- CN105012333B CN105012333B CN201510436403.5A CN201510436403A CN105012333B CN 105012333 B CN105012333 B CN 105012333B CN 201510436403 A CN201510436403 A CN 201510436403A CN 105012333 B CN105012333 B CN 105012333B
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Abstract
The invention belongs to biopharmaceutical technology, a kind of efficient anti-inflammatory PB ODN sequence and its application are specifically disclosed, for the sequence as shown in SEQ ID NO:1, which has efficient anti-inflammatory effect；Preparation containing the sequence is very beneficial for controlling pig disease and inflammation, substitution or part substitute antibiotics can be successfully realized, to reduce the usage amount of antibiotic and vaccine；Keep animal based food safer, meet environmentally protective requirement, advantageously reduce the cost of cultivated animals manufacturing enterprise, can also improve the breeding efficiency of breeding enterprise.
Description
Technical field
The present invention relates to biopharmaceutical technology, more particularly, to a kind of efficient anti-inflammatory PB ODN sequence and its
Using.
Background technique
Numerous domestic anti-inflammatory drugs for animals all rely primarily on antibiotic, but antibiotic is used for a long time, and will lead to serious
Drug resistance, lot of domestic and foreign enterprises has begun limitation using Multiple Classes of Antibiotics at present, and also high degree limits the abuse of antibiotic
The outlet of the China Zhi Liao meat product.The means of another prevention veterinary disease are exactly to utilize vaccine.But a variety of pathogens
Serotype is numerous, vaccine cross reaction, and protecting effect is poor.Therefore it is badly in need of a kind of anti-inflammatory preparation of new type of safe.
Probiotics DNA is the main effects factor that probiotics is able to suppress various inflammatory reactions, wherein there is specific benefit
Raw motif (Probiotics Motif).In probiotics DNA the screening of prebiotic motif with screened from e. coli dna before this
CpG motif has the place that plays the same tune on different musical instruments.Here, we are in order to distinguish its inhibition oligonucleotides with mammal source
(Suppressive oligodeoxynucleotide, Sup ODN), is named as prebiotic ODN for the prebiotic motif of probiotics DNA
(Probiotics oligodeoxynucleotide, PB ODN).DNA of bacteria can be acted on by non-methylated CpG sequences
The TLR9 receptor of host immune system, promotes B cell proliferation, induces strong Th1 inflammatory reaction, enhancement antigen antibody response,
Therefore, DNA of bacteria early stage is also applied to treatment of infection.Later, Krieg etc. filtered out the CpG sequence (purine-in DNA of bacteria
Purine-CpG- pyrimidine-pyrimidine), and then the artificial synthesized few tool picodna (CpG containing CpG sequence
Oligodeoxynucleotide, CpG ODN), and confirm that immune effect is significantly larger than DNA of bacteria, it is pre- thus to have started CpG
The brilliant history of bacteriological protection infection and treating cancer.
Similar, 2000, Pisetsky etc. had found that the DNA of mammal has immune suppression function.Then it finds
Sup ODN sequence, the mammals conservative region such as recent mankind's microsatellite DNA and mitochondrial DNA all become new Sup
The upsurge that inhibition ODN is studied has been raised from this in the research site of ODN.
2012, Bouladoux discovery Sup motif was largely present in probiotics DNA, and thought many before this related
What research of the probiotics DNA in terms of inflammation treatment may finally work is exactly Sup motif present in DNA.Think in benefit
There are a kind of completely new immune modulatory motifs in raw bacterium DNA, and there is regulation inflammatory reaction, restores immune microenvironment balance
Function, applicant have carried out a large amount of screenings to probiotics DNA and have analyzed work, what discovery was wherein characterized in the presence of similar " ACTTGG "
The PB ODN motif of high-repetition-rate, by verifying, determining has apparent inflammation ability of regulation and control.Therefore, it is sieved from probiotics DNA
It selects and inhibits movable characteristic sequence with obvious inflammation, can not only be studied for the development and utilization of probiotics and more letters are provided
Breath also can control inflammation after multiple pathogens infection, have considerable Research Prospects and value；But there is presently no PB
The pertinent literature of ODN and report for preventing and treating pig inflammation.
Summary of the invention
The technical problem to be solved by the present invention is to overcome drawbacks described above of the existing technology, a kind of highly effective and safe is provided
Anti-inflammatory PB ODN sequence.
The present invention is that second purpose is to provide the PB ODN preparation containing above-mentioned PB ODN sequence, is based on the preparation,
It can successfully substitute or part substitute antibiotics, avoid the incomplete purpose of vaccine protecting effect.
Third object of the present invention is to provide the applications of above-mentioned PB ODN preparation.
The purpose of the present invention is what is be achieved by the following technical programs:
A kind of efficiently anti-inflammatory PB ODN sequence, as shown in SEQ ID NO:1.The present invention is screened from probiotics DNA and is obtained
A kind of PB ODN sequence was obtained, which has efficient anti-inflammatory effect.
PB ODN sequence preparation method of the present invention is as follows: passing through the guarantor to most Escherichia coli and Bacillus acidi lactici
It keeps repetitive sequence to be analyzed, discovery " CCAAGG " and " CCAAGC " may be the potential inhibitory motifs of two classes, and reference
The method of Bouladoux, obtained from ncbi database the genome of 28 kinds of Bacillus acidi lacticis, the genome of 27 kinds of Escherichia coli,
Nearly 100 genomes such as staphylococcus aureus gene group, pig microsatellite DNA, pig mtdna and pig Telomerase DNA, by
" fuzznuc " on-line analysis software analyzes the copy number of above-mentioned motif, refers again to the screening side Hu great Li etc. (Hu great Li etc., 2007)
Method carries out ODN screening active ingredients and obtains.
Before use, PB ODN sequence of the present invention need to pass through thioated, thioated processing is this field routine techniques.
The present invention also provides the preparations (referred to as PB ODN preparation) for containing above-mentioned PB ODN sequence；Specifically, can incite somebody to action
PB ODN sequence after thioated is 10~100 μ g/ml with sterile PBS solution compound concentration, is stored in -15~-25 DEG C.
Preferably, the composition of the PBS solution are as follows: in percentage by weight, 0.6~1.0% NaCl, 0.01~
0.04% KCl, 0.0124~0.0164% Na2HPO4, 0.014~0.034% KH2PO4, at 115~121 DEG C, sterilizing 15~
25min。
Specifically, the sterilizing can use damp and hot autoclaving, and operation is referred to this field routine techniques.
The present invention also provides application of the PB ODN sequence in the preparation that preparation treats or prevents pig disease.
It is highly preferred that the pig disease is the disease as caused by haemophilus suis.
The present invention also provides above-mentioned preparations to treat and/or prevent the application in pig disease, specifically, when for treating
When, it is 0.2~1ml PB ODN preparation to be subcutaneously injected by per kilogram pig weight immediately, after 3~7 days after the disease incidence of pig
It is administered again；It is disease incidence peak period the last week in pig when being used to prevent, is subcutaneously injected by per kilogram animal weight
0.1~0.5ml PB ODN preparation, is administered again after 3~7 days.
Compared with prior art, the invention has the following advantages:
The present invention provides a kind of efficiently anti-inflammatory PB ODN sequence and its application, which has efficient anti-inflammatory effect；
Preparation containing the sequence is very beneficial for controlling pig disease and inflammation, can successfully substitute or part substitute antibiotics, to drop
The usage amount of low antibiotic and vaccine；Keep animal based food safer, meet environmentally protective requirement, advantageously reduces cultivation
The cost of animal productiong enterprise can also improve the breeding efficiency of breeding enterprise.
Specific embodiment
The contents of the present invention are further illustrated combined with specific embodiments below, but should not be construed as limiting the invention.
Without departing from the spirit and substance of the case in the present invention, to simple modifications or substitutions made by the method for the present invention, step or condition,
It all belongs to the scope of the present invention；Unless otherwise specified, technological means used in embodiment is well known to those skilled in the art
Conventional means.
Embodiment 1
The sequence (SEQ ID NO:1) of PB ODN: existing gene work can be used in 5 '-TCCAAGTACTTGGACTTGG-3 '
Journey is chemically synthesized, the sequence through thioated handle (thioated processing can with reference to this field common process into
Row).After synthesis, the preparation (PB ODN preparation) of 10 μ g/ml is configured to sterile PBS solution, be stored in -15 DEG C it is spare.
PBS solution composition are as follows: in percentage by weight, 0.6% NaCl, 0.01% KCl, 0.0124%
Na2HPO4, 0.014% KH2PO4It is soluble in water；Then using damp and hot autoclaving at 115 DEG C, sterilize 20min.It is damp and hot
Autoclaving is sterilizing methods commonly used in the art, and operation is referred to prior art progress.
Prevention: in disease incidence peak period the last week of pig, by PB described in per kilogram animal weight subcutaneous injection 0.1ml
ODN preparation is administered again after 3 days.
Treatment: the disease of pig after the onset of, immediately by per kilogram animal weight subcutaneous injection 0.2ml described in PB ODN system
Agent is administered again after 3 days.
Embodiment 2
The sequence (SEQ ID NO:1) of PB ODN: existing gene work can be used in 5 '-TCCAAGTACTTGGACTTGG-3 '
Journey is chemically synthesized, the sequence through thioated handle (thioated processing can with reference to this field common process into
Row).After synthesis, the preparation (PB ODN preparation) of 30 μ g/ml is configured to sterile PBS solution, it is standby at a temperature of being stored in -20 DEG C
With.
PBS solution composition are as follows: in percentage by weight, 0.7% NaCl, 0.02% KCl, 0.0134%
Na2HPO4, 0.024% KH2PO4It is soluble in water；Then using damp and hot autoclaving at 117 DEG C, sterilize 21min.It is damp and hot
Autoclaving is sterilizing methods commonly used in the art, and operation is referred to prior art progress.
Prevention: in disease incidence peak period the last week of pig, by PB described in per kilogram animal weight subcutaneous injection 0.2ml
ODN preparation is administered again after 4 days.
Treatment: the disease of pig after the onset of, immediately by per kilogram animal weight subcutaneous injection 0.3ml described in PB ODN system
Agent is administered again after 4 days.
Embodiment 3
The sequence (SEQ ID NO:1) of PB ODN: existing gene work can be used in 5 '-TCCAAGTACTTGGACTTGG-3 '
Journey is chemically synthesized, the sequence through thioated handle (thioated processing can with reference to this field common process into
Row).After synthesis, the preparation (PB ODN preparation) of 50 μ g/ml is configured to sterile PBS solution, it is standby at a temperature of being stored in -22 DEG C
With.
PBS solution composition are as follows: in percentage by weight, 0.8% NaCl, 0.03% KCl, 0.0144%
Na2HPO4, 0.0245% KH2PO4It is soluble in water；Then using damp and hot autoclaving at 118 DEG C, sterilize 22min.It is damp and hot
Autoclaving is sterilizing methods commonly used in the art, and operation is referred to prior art progress.
Prevention: in disease incidence peak period the last week of pig, by PB described in per kilogram animal weight subcutaneous injection 0.3ml
ODN preparation is administered again after 5 days.
Treatment: the disease of pig after the onset of, immediately by per kilogram animal weight subcutaneous injection 0.4ml described in PB ODN system
Agent is administered again after 5 days.
Embodiment 4
The sequence (SEQ ID NO:1) of PB ODN: existing gene work can be used in 5 '-TCCAAGTACTTGGACTTGG-3 '
Journey is chemically synthesized, the sequence through thioated handle (thioated processing can with reference to this field common process into
Row).After synthesis, the preparation (PB ODN preparation) of 100 μ g/ml is configured to sterile PBS solution, it is standby at a temperature of being stored in -25 DEG C
With.
PBS solution composition are as follows: in percentage by weight, 1.0% NaCl, 0.04% KCl, 0.0164%
Na2HPO4, 0.034% KH2PO4It is soluble in water；Then using damp and hot autoclaving at 121 DEG C, sterilize 25min.It is damp and hot
Autoclaving is sterilizing methods commonly used in the art, and operation is referred to prior art progress.
Prevention: in disease incidence peak period the last week of pig, by PB described in per kilogram animal weight subcutaneous injection 0.5ml
ODN preparation is administered again after 7 days.
Treatment: the disease of pig after the onset of, immediately by per kilogram animal weight subcutaneous injection 0.5ml described in PB ODN system
Agent is administered again after 7 days.
Comparative example 1
Experimental method uniquely the difference is that, is substituted the partial sequence of PB ODN used, compares ODN1 with embodiment 4
Sequence is as follows: 5 '-TCCACTGTGCTTGGACTTGG-3 '.
Comparative example 2
Experimental method uniquely the difference is that, is substituted the partial sequence of PB ODN used, compares ODN2 with embodiment 4
Sequence is as follows: 5 '-TCCAAGTACTAGGTGTTGG-3 '.
Comparative example 3
With embodiment 4 uniquely the difference is that, the part sequence of PB ODN used is substituted in experimental method --- comparison ODN3
Sequence is as follows: 5 '-TCGCACTTGATGCTTGG-3 '.
5 cytologic experiment of embodiment
With the peripheral blood lymphocytes (PBMC) of 16 age in days pigs (great Bai × length is white) for experimental cell, the present invention is respectively adopted
The PB ODN preparation that Examples 1 to 4 and 1~3 the method for comparative example are prepared is as prevention and treatment preparation, haemophilus suis conduct
Pathogen (HPS, ATCC19417), using PBS as blank control, haemophilus parasuis vaccine before existing commercially available Wuhan section
(HPSV) it is used as experiment vaccine, using veterinary antibiotic (Ampicillin) as experiment antibiotic, to compare ODN1,2,3 groups
As ODN effect comparison, the expression of inflammatory factor IL-1 β, IL-18 are analyzed, the results are shown in Table 1.PB ODN and HPS is co-cultured
Swine PBMC s, discovery can significantly lower the expression of inflammatory factor IL-1 β, IL-18, and antibiotic group can also lower inflammatory factor
Expression, but it is good not as good as the effect of PB ODN, and comparison ODN1,2,3 groups and vaccine are little to the effect of inflammatory factor, and the above results are said
Bright PB ODN has the effect of anti-HPS infection.
6 zoopery of embodiment
With 16 age in days pigs (great Bai × length is white) for experimental subjects, haemophilus parasuis vaccine before existing commercially available Wuhan section
(HPSV) it is used as experiment vaccine, haemophilus suis is as pathogen (HPS, ATCC19417), using PBS as blank control, point
Not Cai Yong the PB ODN preparation that is prepared of 1~4 the method for the embodiment of the present invention as prevention and treatment preparation, with veterinary antibiotic
(Ampicillin) is used as experiment antibiotic.PB ODN preparation is immunized in HPS infection the last week, as preventive effect research；?
PB ODN preparation is immunized immediately after HPS infection, as therapeutic effect research.Disease incidence was observed respectively at immune 14th day, was tested
As a result such as table 2, shown in table 3.
From table 2~3 it is found that the PB ODN preparation prepared through the method for the present invention, effect is suitable with antibiotic in terms of prevention,
It is got well than the effect of vaccine.Effect is better than antibiotic outline in terms for the treatment of, more preferable than the effect of vaccine.Therefore, PB ODN system
Agent is hopeful to replace completely or part replaces conventional antibiotic, dynamic to cultivation as a kind of novel, efficient, safe formulation application
In the diseases prevention of object.
7 toxicity research of embodiment
The present embodiment selects 7 age in days balb/c mouse, is randomly divided into blank control group and by 1~4 institute of the embodiment of the present invention
The PB ODN preparation group that the method for stating is prepared, each animal injecting immune 0.5ml, (every group of 3 repetitions).Control group fed is adopted
With the methods of indices such as feeding experiment, animal tissue's dissection and appearance property.Test period is 2 months, when off-test
Detect the death rate and lesion tissue situation of mouse.As the result is shown (table 4): the mortality of animals of PB ODN processing group and control group
It is zero, anatomic tissue does not find obvious lesion, and processing group mouse is vivaciously active, illustrates PB ODN to BALB/C mice safety nothing
It poisons.
SEQUENCE LISTING
<110>Agricultural University Of South China
<120>a kind of efficient anti-inflammatory PB ODN sequence and its application
<130>
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213>sequence of PB ODN
<400> 1
tccaagtact tggacttgg 19
<210> 2
<211> 20
<212> DNA
<213>ODN1 sequence
<400> 2
tccactgtgc ttggacttgg 20
<210> 3
<211> 19
<212> DNA
<213>ODN2 sequence
<400> 3
tccaagtact aggtgttgg 19
<210> 4
<211> 17
<212> DNA
<213>ODN3 sequence
<400> 4
tcgcacttga tgcttgg 17
Claims (6)
1. a kind of anti-inflammatory PB ODN sequence, which is characterized in that sequence is as shown in SEQ ID NO:1.
2. PB ODN sequence according to claim 1, which is characterized in that the PB ODN sequence passes through thioated.
3. the preparation containing PB ODN sequence described in claim 2.
4. preparation according to claim 3, which is characterized in that the PB ODN sequence after thioated is molten with sterile PBS
Liquid compound concentration is 10~100 μ g/ml, is stored in -15~-25 DEG C.
5. preparation according to claim 4, which is characterized in that the composition of the PBS solution are as follows: according to weight percent
Meter, 0.6~1.0% NaCl, 0.01~0.04% KCl, 0.0124~0.0164% Na2HPO4, 0.014~0.034%
KH2PO4, at 115~121 DEG C, sterilize 15~25min.
6. the system of the pig disease as caused by haemophilus suis in preparation treatment and/or prevention of PB ODN sequence described in claim 1
Application in agent.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1926145A (en) * | 2005-01-28 | 2007-03-07 | 权滢周 | Oligonucleotides extracted from mycobacteria for stimulating immune function, treating immune-related diseases, allergic dermatitis and/or protecting normal immune cells |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1926145A (en) * | 2005-01-28 | 2007-03-07 | 权滢周 | Oligonucleotides extracted from mycobacteria for stimulating immune function, treating immune-related diseases, allergic dermatitis and/or protecting normal immune cells |
Non-Patent Citations (1)
| Title |
|---|
| 益生菌防治小儿呼吸系统疾病研究进展;顾美群等;《医学研究杂志》;20140331;第43卷(第3期);第158-160页 |
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